The invention relates to methods for the production of vectors that, after their transfection into eukaryotic cells, are suitable for inhibiting in a targeted manner by means of RNA interference the formation of one or several proteins, and also to such vectors.
One possibility shown recently of inhibiting gene expression is based on the synthesis of double-stranded RNA molecules. Individual genes can be switched off in a targeted manner very effectively and faster using this double-strand RNA (dsRNA) than with any other method, without disrupting the protein formation of neighbouring genes. The underlying principle is termed RNA interference, called RNAi for short. The dsRNA sequence causing this phenomenon is termed siRNA (small interference RNA).
The siRNA does not prevent the gene from being read but switches on a mechanism of the cell, which prevents production of mRNA molecules read off from the gene and thus prevents the formation of the corresponding protein (post-transcriptional gene silencing).
This targeted degradation of mRNA is triggered by short siRNA molecules with a length of 19 to 23 RNA bases, which are homologous to the target mRNA whose transcription into a protein is to be prevented. The siRNA molecules combine with special endoribonucleases to form an RNA protein complex of the cell called “RISC” (RNA-induced silencing complex). As these complexes are built up, the two strands of RNA dissociate, resulting in so-called activated RISCs, which each contain a single strand of the siRNA molecule. Activated RISCs containing the antisense strand that is complementary to the target mRNA bind to the latter and the endoribonuclease of the RNA-protein complex now ensures the sequence-specific degradation of the mRNA.
The siRNA can be created in the cell experimentally or it can be transported in from the outside. This is done using synthetically produced siRNA molecules, which can be administered both in vitro and in vivo.
However, this method has technical limits. Alongside the general instability of the synthetic siRNA in the medium and also in the cell, inhibition using synthetic siRNA is in principle possible only temporarily, and many cells (e.g. neuronal cells) can be transfected only very inefficiently. Studies based on the transfection of synthetic siRNA are therefore limited as a rule both in terms of time, to 1 to 5 days, and in terms of cell type. Furthermore, the high production costs and the lengthy production also represent disadvantages.
The other method is to create siRNA in the cell by means of vectors. These are viral or plasmid-based vectors that only lead to the formation of the siRNA sequences by means of expression once they are in the cell. The advantages compared to transfection with synthetic siRNA are the more stable and more regulated transcription of the corresponding siRNA sequence.
However, the plasmid-based vectors not only exhibit a low transfection efficiency but also have an elaborate production process. It is therefore necessary to select stable clones. This process is often lengthy and can take weeks or even months, with the frequent occurrence of numerous potential difficulties that are inherent to the cloning experiments. For checking the product, sequencing procedures are necessary that are also labour-intensive and costly.
Furthermore, plasmid-based vectors contain antibiotic-resistance genes, which are necessary for their selection. For this reason, such vectors are not suitable for application in living organisms. The possible recombination with ubiquitous bacteria occurring in the organism harbours the risk of increasing occurrence of bacteria resistant to antibiotics. The spread of resistance to antibiotics is a serious problem and such a path would be irresponsible.
As viral vectors are capable of efficient and targeted transfection, they offer an advantage compared to synthetic siRNA molecules and to plasmid-based vectors.
However, there are reservations restricting the use of such viral vectors in therapeutic application. Here, too, the recombination of viral sequences with naturally occurring viruses represents an inherent safety risk, since the creation of new, pathogenic hybrid viruses must be feared. In addition, their production is elaborate and costly.
As even the inhibition of the expression of only one gene product with the use of expression systems is associated with a large number of complications, it is almost axiomatic that simultaneously switching off the expression of several gene products in a cell or a tissue is much more complex and therefore more difficult to accomplish.
One of the main problems with the multiple inhibition of gene expression by RNA interference, when several independent constructs are used, is the low probability that a cell will be transfected by only one construct. This low probability decreases exponentially with the number of constructs used. Often, however, for certain approaches in genetic therapy a controlled, simultaneous inhibition of gene expression is required.
A large number of methods exist that enable the cotranscription of several RNA molecules in a cell. The simplest method is the cotransfection of two independent expression constructs (in the following also called vectors). Another method consists of transfection with a vector carrying the two independent expression cassettes. Such constructs can also be used for the synthesis of siRNA molecules.
With both possibilities there is however the danger that the transcripts differ significantly in relation to their number, processing, half-lives and translational efficiency and therefore in the quantity of the protein expressed, with the result that their use is characterised by ineffectiveness and poor reproducibility. Therefore the use of such expression systems would also lead to siRNA transcription of varying degrees, which would ultimately result in the unequal inhibition of the relevant genes.
The construction of dicistronic and polycistronic RNA molecules with the use of IRES (internal ribosome entry site) elements represents another possibility for the coexpression of several genes. The latter allow the initiation of the translation by ribosomes independently of the mRNA cap structure by sequence elements. For the first time IRES elements have been discovered in the mRNA of the picornavirus (Pelletier and Sonenberg, 1988, Nature 334: 320-325, Jang et al., 1988, Journal Virology 62: 2636-2643).
For the construction of bicistronic vectors, most frequently the IRES elements of the poliovirus and EMCV (encephalomyocarditis virus) are used (Dirks et al., 1993, Gene 128: 247-249). Unfortunately their efficiency varies strongly depending on the cell line used (Borman et al., 1997, Nucleic Acids Res. 25: 925-932).
Most of the available bicistronic expression cassettes consist of a selectable marker or a reporter gene, which are arranged on the 3′-side of the IRES element and an MCS (multiple cloning site) for the insertion of the desired gene (Dirks et al., 1993, Gene 128: 247-249). Tricistronic and polycistronic expression systems with the use of IRES elements are also known (Zitvogel et al., 1994, Hum Gene Ther 5: 1493-1506, Fussenegger et al., 1998a, Nature Biotechnol. 16: 468-472, Mielke et al., 2000, Gene 254. 1-8).
However, there are reservations restricting the use of viral IRES elements in therapeutic application. Here, too, the recombination of viral sequences with naturally occurring viruses represents an inherent safety risk, since the creation of new, pathogenic hybrid viruses must be feared.
Multicistronic vectors can also be constructed by linking transcription units without IRES elements. On the one hand, the desired genes can be cloned via various MCS into a plasmid vector, and on the other, expression cassettes isolated from plasmids can be connected by DNA linkers to form linear multicistronic vectors (sang et al., 1997, Bio Techniques 22: 68).
However, investigations of the expression rates of linear cis-linked genes have shown a strong negative influence on the transcription of expression cassettes arranged in this manner (Esperet et al., 2000, J Biol Chem 275: 25831-25839).
Plasmid vectors for the expression of multiple genes differ from the conventional plasmid vectors in the number of cloned transcription units. In this context promoters or poly(A) sequences of identical or different origin can be used for the transcription units.
The disadvantage that arises from the use of differing promoters is that the promoters differ in their strength and therefore the expression rates of the individual genes can vary, yet the use of identical promoters can lead to formation of secondary DNA structures, which can lead to loss of function of the promoters as a consequence.
All the mentioned vectors for multiple gene expression have in common that they are based either on plasmids or on viral vectors. Alongside all the shortcomings described of the current multiple-coding vectors, in addition they exhibit the disadvantages of this type of vector. The disadvantages of viral vectors, which include instability of the attenuated vaccination strain, and the shortcomings of plasmid DNA vectors, such as the spread of resistance genes against antibiotics that accompanies their use (described in detail in EP 0 941 318 B1), are sufficiently well known.
Therefore it would be desirable to have expression systems for siRNA that are also in a position to inhibit the gene expression of several genes simultaneously. Probably the greatest problem in resolving this question is a linker connecting the sequences to create the siRNA molecules, which in each case ensures sufficient transcription for inhibition of gene expression of these sequences.